This A1 competitive renewal of GM088276 is a multidisciplinary collaboration to investigate the biosynthesis of diphthamide, a unique protein posttranslational modification that occurs on archaeal and eukaryotic translation elongation factor 2. This modification has been known for over 30 years and is the target of several bacterial toxins, including diphtheria toxin. However, the biosynthesis and biological function are still poorly understood. Previous studies by others suggest that there are five proteins (Dph1-5) required for the first two steps of the biosynthesis, while no proteins were identified for the thid (and last) amidation step. Interestingly, deletion of several of the biosynthesis genes is found in tumors. With previous grant support, we made a number of important findings, including the discovery of a novel radical SAM enzyme for the first step of diphthamide biosynthesis and the identification of two new proteins required for the last step of the biosynthesis. This renewal wil build on these finding to further understand the chemistry and enzymology of diphthamide biosynthesis and to elucidate the complete biosynthetic pathway for the first time. The interesting Fe-S enzyme chemistry that will be elucidated in this proposal will significantly expand the chemistry scope and mechanistic understanding of Fe-S enzymes. The understanding of diphthamide biosynthesis will help to understand the biological functions of diphthamide and why deletion of diphthamide biosynthesis genes promotes tumorigenesis.